The poxviridae comprise a large family of complex DNA viruses that replicate in the cytoplasm of vertebrate and invertebrate cells. The family of poxviridae can be divided into the subfamily chordopoxvirinae (vertebrate poxviruses) and entomopoxvirinae (insect poxviruses) (Fields Virology/eds.: Fields, B. N., Knipe, D. M., Howley, P. M.; 3rd ed/ISBN 0-7817-0253-4/ see in particular chapter 83).
The chordopoxvirinae comprise numerous animal poxviruses (classified in different genera), such as camelpox-viruses, sheeppox-virus, goatpox-virus or Avipoxviruses, in particular fowlpoxvirus and also poxvirusus that are of relevance for humans such as the variola virus and the vaccinia virus.
Pox-viruses, in particular chordopoxvirinae, are important pathogens in humans and animals. There is also a long history of vaccination against pox-virus infections. Nearly two centuries ago, humans were prophylactically inoculated with cowpox to immunise them against smallpox. Later immunisation was performed with the Vaccinia virus. However, smallpox vaccination with this Vaccinia virus resulted occasionally in serious complications, such as postvaccinal encephalitis, generalised Vaccinia or contact infection. Then, a new vaccine that does not show these complications, was developed by Anton Mayr. The pox vaccine consists of the poxvirus Modified Vaccinia Virus Ankara (MVA) and was used for vaccination against smallpox in about 150 000 vaccinations without causing any complications related to the vaccination. Even children with immunologic deficiencies did not show serious side effects. The MVA was obtained by mutation and selection of the original vaccinia virus Ankara after 575 passages in chicken embryo fibroblast cultures. The safety of this MVA is reflected by biological, chemical and physical characteristics. MVA has a reduced molecular weight, six deletions in the genome, and is highly attenuated for mammalian cells, i.e., DNA and protein is synthesised but virtually no viral particles are produced.
The vaccination against smallpox was highly successful. In 1979, the World Health Organisation declared the eradication of smallpox. Accordingly, the mass vaccination of children was discontinued and only laboratory workers and members of the armed forces of some countries are vaccinated.
With the eradication of smallpox, the predominant cause of pox viral infection in humans was removed. However, some non-human poxviruses have reduced host specificity, i.e., they cause infections not only in their typical host (e.g. for cowpox the cow), but also in other animals, (e.g. rats and cats). Humans can be infected by this route as well. Since parts of the population are no longer immune against smallpox, poxvirus infections of animal species can be dangerous for them. Domestic animals are the main source of infection for humans. Accordingly, the vaccination of domestic animals against poxviruses is. of increasing importance. In addition, poxviruses are important vectors for the expression of foreign genes for example for use as a vaccine or for gene therapy, i.e. to transfer nucleic acid sequences into a target cell where they are expressed. Consequently, an efficient and cost effective production method for poxviruses is required.
Poxviruses can be amplified in different cell types. For example, chordopoxvirinae, in particular MVA are amplified in cell cultures of primary or secondary chicken embryo fibroblasts (CEF). The cells are obtained from embryos of chicken eggs that are incubated for 10 to 12 days. The cells of the embryos are then dissociated and purified. These primary CEF cells can either be used directly or after one further cell passage as secondary CEF cells. Subsequently, the primary or secondary CEF cells are infected with the MVA. For the amplification of MVA the infected cells are incubated for 2-3 days at 37° C. (see, e.g., Meyer, H. et al. 1991; J. of General Virology 72, 1031-1038; Sutter et al. 1994, Vaccine, Vol. 12, No. 11, 1032-1040). Although other chordopoxviruses are amplified in different cell types, the same temperature of 37° C. is chosen in those cases. For example, the Vaccinia virus obtainable from ATCC (No. VR1354), which is cultivated in HeLa S3 cells (human cervix carcinoma cells) is also incubated for 3 days at 37° C. (Current protocols in molecular biology 1998, Chapter 16, Unit 16.16, John Wiley & Sons, Inc). Furthermore, the MVA adapted for growing in Vero cells (monkey kidney cells) is also amplified at 37° C. (PCT/EP01/02703). Consequently, independent from the cells used for amplification and independent form the species or strain of the chordopoxvirus, amplification of the viruses is performed at 37° C. This selected temperature corresponds well with the general knowledge of the skilled practitioner: Pox-viruses nearly exclusively amplified in the laboratories are obtained from warm-blooded animals with a body temperature of approximately 37° C. Since chordopoxviruses are adapted for growing in said animals, they are adapted for growing at 37° C., i.e. they should amplify most efficiently at 37° C.
Because of similar reasons Entomopoxviruses are cultivated at temperatures lower than 37° C.: The body temperature of insects is significantly lower than 37° C. and depends to a larger extent on the temperature of the environment. Thus, in contrast to Chordopoxviruses the Entomopoxviruses are adapted for growing at lower temperatures. U.S. Pat. No. 5,721,352 and U.S. Pat. No. 5,174,993 disclose an optimal temperature for growth of the Entomopoxvirus species Amsacta moorei Entomopoxvirus (AmEPV) of 28° C. in the laboratory. However, these patents do not disclose the cultivation of Chordopoxviruses under these temperature conditions.
Furthermore, production of vaccines against other viral infections is in general performed at 37° C. Only some measles vaccines are produced at a lower temperature. In this case, a measles vaccine, which was originally produced at 37° C. and which frequently caused severe side effects, was attenuated by continuous passaging of the virus at 32° C. After 85 passages of the strain at 32° C. the strain was attenuated, i.e. the disease-causing capacity of the virus was considerably reduced (Plotkin, Orenstein: Vaccines, 3rd edition, 230-232). In conclusion, viruses of warm-blooded animals and particularly Vaccinia viruses are expected to amplify most efficiently at 37° C., since they are found in animals with said body temperature and adaptation to a lower temperature is only achieved after multiple passages at said lower temperature. Furthermore, adaptation to a lower temperature is associated with attenuation and therefore often with reduced reproduction capacity of the virus.
U.S. Pat. No. 5,616,487 discloses a process for producing a stabilized virus, in particular a stabilized retrovirus, by culturing virus producing cells with a stabilizing agent at a temperature below 37° C. The stabilizing agents are lipids or surfactants. The patent specifically discloses Pluronic F-68 and Lipid Concentrate as stabilizing agents. Lipid Concentrate is said to contain cholesterol, cod liver oil, Pluronic F-68, d-alpha-tocopherol acetate and Tween 80. In an alternative embodiment U.S. Pat. No. 5,616,487 discloses a process for cultivating specific retrovirus producing cells at a temperature of lower than 37° C., wherein the produced retrovirus is stabilized using a stabilizer as defined above.